Proteogenomics of clear cell renal cell carcinoma response to tyrosine kinase inhibitor.

The tyrosine kinase inhibitor (TKI) Sunitinib is one the therapies approved for advanced renal cell carcinoma. Here, we undertake proteogenomic profiling of 115 tumors from patients with clear cell renal cell carcinoma (ccRCC) undergoing Sunitinib treatment and reveal the molecular basis of differential clinical outcomes with TKI therapy. We find that chromosome 7q gain-induced mTOR signaling activation is associated with poor therapeutic outcomes with Sunitinib treatment, whereas the aristolochic acid signature and VHL mutation synergistically caused enhanced glycolysis is correlated with better prognosis. The proteomic and phosphoproteomic analysis further highlights the responsibility of mTOR signaling for non-response to Sunitinib. Immune landscape characterization reveals diverse tumor microenvironment subsets in ccRCC. Finally, we construct a multi-omics classifier that can detect responder and non-responder patients (receiver operating characteristic-area under the curve, 0.98). Our study highlights associations between ccRCC molecular characteristics and the response to TKI, which can facilitate future improvement of therapeutic responses.

METTL3 boosts mitochondrial fission and induces cardiac fibrosis by enhancing LncRNA GAS5 methylation.

Dysregulated mitochondrial metabolism occurs in several pathological processes characterized by cell proliferation and migration. Nonetheless, the role of mitochondrial fission is not well appreciated in cardiac fibrosis, which is accompanied by enhanced fibroblast proliferation and migration. We investigated the causes and consequences of mitochondrial fission in cardiac fibrosis using cultured cells, animal models, and clinical samples. Increased METTL3 expression caused excessive mitochondrial fission, resulting in the proliferation and migration of cardiac fibroblasts that lead to cardiac fibrosis. Knockdown of METTL3 suppressed mitochondrial fission, inhibiting fibroblast proliferation and migration for ameliorating cardiac fibrosis. Elevated METTL3 and N6-methyladenosine (m6A) levels were associated with low expression of long non-coding RNA GAS5. Mechanistically, METTL3-mediated m6A methylation of GAS5 induced its degradation, dependent of YTHDF2. GAS5 could interact with mitochondrial fission marker Drp1 directly; overexpression of GAS5 suppressed Drp1-mediated mitochondrial fission, inhibiting cardiac fibroblast proliferation and migration. Knockdown of GAS5 produced the opposite effect. Clinically, increased METTL3 and YTHDF2 levels corresponded with decreased GAS5 expression, increased m6A mRNA content and mitochondrial fission, and increased cardiac fibrosis in human heart tissue with atrial fibrillation. We describe a novel mechanism wherein METTL3 boosts mitochondrial fission, cardiac fibroblast proliferation, and fibroblast migration: METTL3 catalyzes m6A methylation of GAS5 methylation in a YTHDF2-dependent manner. Our findings provide insight into the development of preventative measures for cardiac fibrosis.

Dysregulated Gln-Glu-α-ketoglutarate axis impairs maternal decidualization and increases the risk of recurrent spontaneous miscarriage.

Recurrent spontaneous miscarriage (RSM) affects 1%-2% of fertile women worldwide and poses a risk of future pregnancy complications. Increasing evidence has indicated that defective endometrial stromal decidualization is a potential cause of RSM. Here, we perform liquid chromatography with mass spectrometry (LC-MS)-based metabolite profiling in human endometrial stromal cells (ESCs) and differentiated ESCs (DESCs) and find that accumulated α-ketoglutarate (αKG) derived from activated glutaminolysis contributes to maternal decidualization. Contrarily, ESCs obtained from patients with RSM show glutaminolysis blockade and aberrant decidualization. We further find that enhanced Gln-Glu-αKG flux decreases histone methylation and supports ATP production during decidualization. In vivo, feeding mice a Glu-free diet leads to a reduction of αKG, impaired decidualization, and an increase of fetal loss rate. Isotopic tracing approaches demonstrate Gln-dependent oxidative metabolism as a prevalent direction during decidualization. Our results demonstrate an essential prerequisite of Gln-Glu-αKG flux to regulate maternal decidualization, suggesting αKG supplementation as a putative strategy to rectify deficient decidualization in patients with RSM.

Comprehensive proteogenomic characterization of early duodenal cancer reveals the carcinogenesis tracks of different subtypes.

The subtypes of duodenal cancer (DC) are complicated and the carcinogenesis process is not well characterized. We present comprehensive characterization of 438 samples from 156 DC patients, covering 2 major and 5 rare subtypes. Proteogenomics reveals LYN amplification at the chromosome 8q gain functioned in the transmit from intraepithelial neoplasia phase to infiltration tumor phase via MAPK signaling, and illustrates the DST mutation improves mTOR signaling in the duodenal adenocarcinoma stage. Proteome-based analysis elucidates stage-specific molecular characterizations and carcinogenesis tracks, and defines the cancer-driving waves of the adenocarcinoma and Brunner's gland subtypes. The drug-targetable alanyl-tRNA synthetase (AARS1) in the high tumor mutation burden/immune infiltration is significantly enhanced in DC progression, and catalyzes the lysine-alanylation of poly-ADP-ribose polymerases (PARP1), which decreases the apoptosis of cancer cells, eventually promoting cell proliferation and tumorigenesis. We assess the proteogenomic landscape of early DC, and provide insights into the molecular features corresponding therapeutic targets.

Integrative proteogenomic characterization of early esophageal cancer.

Esophageal squamous cell carcinoma (ESCC) is malignant while the carcinogenesis is still unclear. Here, we perform a comprehensive multi-omics analysis of 786 trace-tumor-samples from 154 ESCC patients, covering 9 histopathological stages and 3 phases. Proteogenomics elucidates cancer-driving waves in ESCC progression, and reveals the molecular characterization of alcohol drinking habit associated signatures. We discover chromosome 3q gain functions in the transmit from nontumor to intraepithelial neoplasia phases, and find TP53 mutation enhances DNA replication in intraepithelial neoplasia phase. The mutations of AKAP9 and MCAF1 upregulate glycolysis and Wnt signaling, respectively, in advanced-stage ESCC phase. Six major tracks related to different clinical features during ESCC progression are identified, which is validated by an independent cohort with another 256 samples. Hyperphosphorylated phosphoglycerate kinase 1 (PGK1, S203) is considered as a drug target in ESCC progression. This study provides insight into the understanding of ESCC molecular mechanism and the development of therapeutic targets.

Identification of prognostic and therapeutic biomarkers in type 2 papillary renal cell carcinoma.

BACKGROUND: Papillary renal cell carcinoma (PRCC) can be divided into type 1 (PRCC1) and type 2 (PRCC2) and PRCC2 share a more invasive phenotype and worse prognosis. This study aims to identify potential prognostic and therapeutic biomarkers in PRCC2. METHODS: A cohort from The Cancer Genome Atlas and two datasets from Gene Expression Omnibus were examined. Common differentially expressed genes (DEGs) were screened and potential biomarkers were explored by using Kaplan-Meier method and cox regression analysis. Functional enrichment analysis was utilized to evaluate the potential biological functions. Tumor infiltrating immune cells were estimated by CIBERSORT algorithm. Ninety-two PRCC2 samples from Fudan University Shanghai Cancer Center were obtained, and immunostaining was performed to validate prognostic and therapeutic significance of the potential biomarker. RESULTS: PRCC2 has worse overall survival and shares distinct molecular characteristics from PRCC1. There was significant higher expression level of Targeting protein for Xklp2 (TPX2) in PRCC2 compared with normal tissues. Higher expression level of TPX2 was significantly associated with worse overall survival in PRCC2 and kinesin family genes expression were found significantly elevated in high risk PRCC2. Abundance of tumor infiltrating M1 macrophage was significantly higher in PRCC2 and it was also associated with worse overall survival. In the FUSCC cohort, higher TPX2 expression was significantly correlated with worse overall and progression-free survival. Retrospective analysis indicated that mTOR inhibitor (everolimus) had greater efficacy in the high-risk group than in the low-risk group (overall response rate: 28.6% vs. 16.7%) and that everolimus had greater efficacy than sunitinib in the high-risk group (overall response rate: 28.6% vs. 20%). CONCLUSIONS: TPX2 was a prognostic and therapeutic biomarker in PRCC2. Higher abundance of tumor infiltrating M1 macrophage was significantly associated with worse overall survival in PRCC2. mTOR inhibitors may have good efficacy in patients with high-risk PRCC2.

Gestational palmitic acid suppresses embryonic GATA-binding protein 4 signaling and causes congenital heart disease.

Dysregulated maternal fatty acid metabolism increases the risk of congenital heart disease (CHD) in offspring with an unknown mechanism, and the effect of folic acid fortification in preventing CHD is controversial. Using gas chromatography coupled to either a flame ionization detector or mass spectrometer (GC-FID/MS) analysis, we find that the palmitic acid (PA) concentration increases significantly in serum samples of pregnant women bearing children with CHD. Feeding pregnant mice with PA increased CHD risk in offspring and cannot be rescued by folic acid supplementation. We further find that PA promotes methionyl-tRNA synthetase (MARS) expression and protein lysine homocysteinylation (K-Hcy) of GATA4 and results in GATA4 inhibition and abnormal heart development. Targeting K-Hcy modification by either genetic ablation of Mars or using N-acetyl-L-cysteine (NAC) decreases CHD onset in high-PA-diet-fed mice. In summary, our work links maternal malnutrition and MARS/K-Hcy with the onset of CHD and provides a potential strategy in preventing CHD by targeting K-Hcy other than folic acid supplementation.

Proteogenomics of diffuse gliomas reveal molecular subtypes associated with specific therapeutic targets and immune-evasion mechanisms.

Diffuse gliomas are devastating brain tumors. Here, we perform a proteogenomic profiling of 213 retrospectively collected glioma tumors. Proteogenomic analysis reveals the downstream biological events leading by EGFR-, IDH1-, TP53-mutations. The comparative analysis illustrates the distinctive features of GBMs and LGGs, indicating CDK2 inhibitor might serve as a promising drug target for GBMs. Further proteogenomic integrative analysis combined with functional experiments highlight the cis-effect of EGFR alterations might lead to glioma tumor cell proliferation through ERK5 medicates nucleotide synthesis process. Proteome-based stratification of gliomas defines 3 proteomic subgroups (S-Ne, S-Pf, S-Im), which could serve as a complement to WHO subtypes, and would provide the essential framework for the utilization of specific targeted therapies for particular glioma subtypes. Immune clustering identifies three immune subtypes with distinctive immune cell types. Further analysis reveals higher EGFR alteration frequencies accounts for elevation of immune check point protein: PD-L1 and CD70 in T-cell infiltrated tumors.

Proteogenomic characterization of cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a highly heterogeneous cancer with limited understanding and few effective therapeutic approaches. We aimed at providing a proteogenomic CCA characterization to inform biological processes and treatment vulnerabilities. APPROACH AND RESULTS: Integrative genomic analysis with functional validation uncovered biological perturbations downstream of driver events including DPCR1 , RBM47 mutations, SH3BGRL2 copy number alterations, and FGFR2 fusions in CCA. Proteomic clustering identified three subtypes with distinct clinical outcomes, molecular features, and potential therapeutics. Phosphoproteomics characterized targetable kinases in CCA, suggesting strategies for effective treatment with CDK and MAPK inhibitors. Patients with CCA with HBV infection showed increased antigen processing and presentation (APC) and T cell infiltration, conferring a favorable prognosis compared with those without HBV infection. The characterization of extrahepatic CCA recommended the feasible application of vascular endothelial-derived growth factor inhibitors. Multiomics profiling presented distinctive molecular characteristics of the large bile duct and the small bile duct of intrahepatic CCA. The immune landscape further revealed diverse tumor immune microenvironments, suggesting immune subtypes C1 and C5 might benefit from immune checkpoint therapy. TCN1 was identified as a potential CCA prognostic biomarker, promoting cell growth by enhancing vitamin B12 metabolism. CONCLUSIONS: We characterized the proteogenomic landscape of 217 CCAs with 197 paired normal adjacent tissues and identified their subtypes and potential therapeutic targets. The multiomics analyses with other databases and some functional validations have indicated strategies regarding the clinical, biological, and therapeutic approaches to the management of CCA.

Proteogenomic characterization of MiT family translocation renal cell carcinoma.

Microphthalmia transcription factor (MiT) family translocation renal cell carcinoma (tRCC) is a rare type of kidney cancer, which is not well characterized. Here we show the comprehensive proteogenomic analysis of tRCC tumors and normal adjacent tissues to elucidate the molecular landscape of this disease. Our study reveals that defective DNA repair plays an important role in tRCC carcinogenesis and progression. Metabolic processes are markedly dysregulated at both the mRNA and protein levels. Proteomic and phosphoproteome data identify mTOR signaling pathway as a potential therapeutic target. Moreover, molecular subtyping and immune infiltration analysis characterize the inter-tumoral heterogeneity of tRCC. Multi-omic integration reveals the dysregulation of cellular processes affected by genomic alterations, including oxidative phosphorylation, autophagy, transcription factor activity, and proteasome function. This study represents a comprehensive proteogenomic analysis of tRCC, providing valuable insights into its biological mechanisms, disease diagnosis, and prognostication.

Single-cell transcriptome profiling of the human endometrium of patients with recurrent implantation failure.

Introduction: Despite great advances in assisted reproductive technology (ART), recurrent implantation failure (RIF) cannot be effectively avoided. Notably, cellular characteristics and communication that regulate endometrial receptivity and differentiation, and its disorders in RIF at window of implantation (WOI) remain rudimentary. Objectives: In this study, we profiled the endometrial cells present at the WOI timing in RIF patients and healthy controls using single-cell RNA sequencing (scRNA-seq) and provided a detailed molecular and cellular map of a healthy and RIF endometrium at the WOI. Method: In the current study, the endometrium from RIF patient (n = 6; age range, 32 - 35 years) and control (Ctrl) (n = 3; age range, 29 - 35 years) groups were studied at a single-cell resolution. single-cell RNA-seq and analysis were performed on the endometrium of patients with RIF and Ctrl. Immunofluorescence, flow cytometry assays, and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to verify cellular identity and function. Results: We profiled the transcriptomes of 60222 primary human endometrial cells isolated from control and RIF patients at a single-cell resolution. We discovered dramatic differential expression of endometrial receptivity-related genes in four major endometrial fibroblast-like cells from RIF patients compared to the control endometrium. We observed that CD49a+CXCR4+NK cells were diminished in proportion with RIF. The decrease in subset of CD63highPGRhigh endometrial epithelial cells with high levels of progesterone receptor, autophagy and exosomes should contribute to the decrease in subset of NK cells. Additionally, we characterized aberrant molecular and cellular characteristics and endometrial cell-cell communication disorders in RIF patients. Conclusion: Our study provides deeper insights into endometrial microenvironment disorder of RIF that are potentially applicable to improving the etiological diagnosis and therapeutics of unexplained RIF.

Proteomic characterization of gastric cancer response to chemotherapy and targeted therapy reveals new therapeutic strategies.

Chemotherapy and targeted therapy are the major treatments for gastric cancer (GC), but drug resistance limits its effectiveness. Here, we profile the proteome of 206 tumor tissues from patients with GC undergoing either chemotherapy or anti-HER2-based therapy. Proteome-based classification reveals four subtypes (G-I-G-IV) related to different clinical and molecular features. MSI-sig high GC patients benefit from docetaxel combination treatment, accompanied by anticancer immune response. Further study reveals patients with high T cell receptor signaling respond to anti-HER2-based therapy; while activation of extracellular matrix/PI3K-AKT pathway impair anti-tumor effect of trastuzumab. We observe CTSE functions as a cell intrinsic enhancer of chemosensitivity of docetaxel, whereas TKTL1 functions as an attenuator. Finally, we develop prognostic models with high accuracy to predict therapeutic response, further validated in an independent validation cohort. This study provides a rich resource for investigating the mechanisms and indicators of chemotherapy and targeted therapy in GC.

Tripartite motif 25 ameliorates doxorubicin-induced cardiotoxicity by degrading p85α.

Doxorubicin (DOX)-based chemotherapy is widely used to treat malignant tumors; however, the cardiotoxicity induced by DOX restricts its clinical usage. A therapeutic dose of DOX can activate ubiquitin-proteasome system. However, whether and how ubiquitin-proteasome system brings out DOX-induced cardiotoxicity remains to be investigated. Here we conducted a proteomics analysis of a DOX-induced cardiotoxicity model to screen the potentially ubiquitination-related molecules. Dysregulated TRIM25 was found to contribute to the cardiotoxicity. In vivo and in vitro cardiotoxicity experiments revealed that TRIM25 ameliorated DOX-induced cardiotoxicity. Electron microscopy and endoplasmic reticulum stress markers revealed that TRIM25 mitigated endoplasmic reticulum stress and apoptosis in DOX-induced cardiomyocytes. Mechanistically, the Co-immunoprecipitation assays and CHX pulse-chase experiment determined that TRIM25 affected p85α stability and promoted its ubiquitination and degradation. This leads to increase of nuclear translocation of XBP-1s, which mitigates endoplasmic reticulum stress. These findings reveal that TRIM25 may have a therapeutic role for DOX-induced cardiotoxicity.

Increased expression of tribbles homolog 3 predicts poor prognosis and correlates with tumor immunity in clear cell renal cell carcinoma: a bioinformatics study.

Tribbles homolog 3 (TRIB3), a pseudokinase that regulates multiple intracellular signaling pathways, has been reported to promote the growth of multiple tumors. However, its role in clear cell renal cell carcinoma (ccRCC) remains unelucidated. We evaluated the role of TRIB3 in ccRCC using publicly available data from The Cancer Genome Atlas and analyzed its relationship with the tumor microenvironment; moreover, we used gene knockout and overexpression techniques to detect the effects of TRIB3 on the biological behavior of ccRCC cells. RT-qPCR and western blotting were used to detect transfection efficiency, and the invasiveness of ccRCC cells was determined by Transwell migration assays. We found that TRIB3 overexpression was significantly associated with increased grade, stage, and distant metastasis, positively correlated with ccRCC invasiveness, and also an independent risk factor for overall survival (OS). In addition, 361 differentially expressed genes (DEGs) related to TRIB3 were identified. Functional enrichment analysis showed that DEGs were mainly enriched in humoral immune responses, collagen-containing extracellular matrix, and serine hydrolase activity. Immune landscape characterization revealed that TRIB3 expression was significantly and negatively associated with CD8+ T and hematopoietic stem cells, whereas it was positively associated with NK T and macrophage M1 cells. Single-cell sequencing showed that localization and binding targets of TRIB3 mainly involved monocytes/macrophages and CD4+ and CD8+ T cells. Overall, our study revealed that elevated TRIB3 expression represents a promising prognostic marker for ccRCC patients and may play a key role in tumor microenvironment modulation.

A proteogenomic analysis of clear cell renal cell carcinoma in a Chinese population.

Clear cell renal cell carcinoma (ccRCC) is a common and aggressive subtype of renal cancer. Here we conduct a comprehensive proteogenomic analysis of 232 tumor and adjacent non-tumor tissue pairs from Chinese ccRCC patients. By comparing with tumor adjacent tissues, we find that ccRCC shows extensive metabolic dysregulation and an enhanced immune response. Molecular subtyping classifies ccRCC tumors into three subtypes (GP1-3), among which the most aggressive GP1 exhibits the strongest immune phenotype, increased metastasis, and metabolic imbalance, linking the multi-omics-derived phenotypes to clinical outcomes of ccRCC. Nicotinamide N-methyltransferase (NNMT), a one-carbon metabolic enzyme, is identified as a potential marker of ccRCC and a drug target for GP1. We demonstrate that NNMT induces DNA-dependent protein kinase catalytic subunit (DNA-PKcs) homocysteinylation, increases DNA repair, and promotes ccRCC tumor growth. This study provides insights into the biological underpinnings and prognosis assessment of ccRCC, revealing targetable metabolic vulnerabilities.

Methylene-bridge tryptophan fatty acylation regulates PI3K-AKT signaling and glucose uptake.

Protein fatty acylation regulates numerous cell signaling pathways. Polyunsaturated fatty acids (PUFAs) exert a plethora of physiological effects, including cell signaling regulation, with underlying mechanisms to be fully understood. Herein, we report that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) regulate PI3K-AKT signaling by modifying PDK1 and AKT2. DHA-administered mice exhibit altered phosphorylation of proteins in signaling pathways. Methylene bridge-containing DHA/EPA acylate δ1 carbon of tryptophan 448/543 in PDK1 and tryptophan 414 in AKT2 via free radical pathway, recruit both the proteins to the cytoplasmic membrane, and activate PI3K signaling and glucose uptake in a tryptophan acylation-dependent but insulin-independent manner in cultured cells and in mice. DHA/EPA deplete cytosolic PDK1 and AKT2 and induce insulin resistance. Akt2 knockout in mice abrogates DHA/EPA-induced PI3K-AKT signaling. Our results identify PUFA's methylene bridge tryptophan acylation, a protein fatty acylation that regulates cell signaling and may underlie multifaceted effects of methylene-bridge-containing PUFAs.

A defective lysophosphatidic acid-autophagy axis increases miscarriage risk by restricting decidual macrophage residence.

Massive infiltrated and enriched decidual macrophages (dMφ) have been widely regarded as important regulators of maternal-fetal immune tolerance and trophoblast invasion, contributing to normal pregnancy. However, the characteristics of metabolic profile and the underlying mechanism of dMφ residence remain largely unknown. Here, we observe that dMφ display an active glycerophospholipid metabolism. The activation of ENPP2-lysophosphatidic acid (LPA) facilitates the adhesion and retention, and M2 differentiation of dMφ during normal pregnancy. Mechanistically, this process is mediated through activation of the LPA receptors (LPAR1 and PPARG/PPARγ)-DDIT4-macroautophagy/autophagy axis, and further upregulation of multiple adhesion factors (e.g., cadherins and selectins) in a CLDN7 (claudin 7)-dependent manner. Additionally, poor trophoblast invasion and placenta development, and a high ratio of embryo loss are observed in Enpp2±, lpar1-/- or PPARG-blocked pregnant mice. Patients with unexplained spontaneous abortion display insufficient autophagy and cell residence of dMφ. In therapeutic studies, supplementation with LPA or the autophagy inducer rapamycin significantly promotes dMφ autophagy and cell residence, and improves embryo resorption in Enpp2± and spontaneous abortion mouse models, which should be dependent on the activation of DDIT4-autophagy-CLDN7-adhesion molecules axis. This observation reveals that inactivation of ENPP2-LPA metabolism and insufficient autophagy of dMφ result in resident obstacle of dMφ and further increase the risk of spontaneous abortion, and provides potential therapeutic strategies to prevent spontaneous abortion.Abbreviations: ACTB: actin beta; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; Atg5: autophagy related 5; ATG13: autophagy related 13; BECN1: beclin 1; CDH1/E-cadherin: cadherin 1; CDH5/VE-cadherin: cadherin 5; CFSE: carboxyfluorescein succinimidyl ester; CLDN7: claudin 7; CSF1/M-CSF: colony stimulating factor 1; CSF2/GM-CSF: colony stimulating factor 2; Ctrl: control; CXCL10/IP-10: chemokine (C-X-C) ligand 10; DDIT4: DNA damage inducible transcript 4; dMφ: decidual macrophage; DSC: decidual stromal cells; ENPP2/ATX: ectonucleotide pyrophosphatase/phosphodiesterase 2; Enpp2±: Enpp2 heterozygous knockout mouse; ENPP2i/PF-8380: ENPP2 inhibitor; EPCAM: epithelial cell adhesion molecule; ESC: endometrial stromal cells; FGF2/b-FGF: fibroblast growth factor 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GPCPD1: glycerophosphocholine phosphodiesterase 1; HE: heterozygote; HIF1A: hypoxia inducible factor 1 subunit alpha; HNF4A: hepatocyte nuclear factor 4 alpha; HO: homozygote; ICAM2: intercellular adhesion molecule 2; IL: interleukin; ITGAV/CD51: integrin subunit alpha V; ITGAM/CD11b: integrin subunit alpha M; ITGAX/CD11b: integrin subunit alpha X; ITGB3/CD61: integrin subunit beta 3; KLRB1/NK1.1: killer cell lectin like receptor B1; KRT7/cytokeratin 7: keratin 7; LPA: lysophosphatidic acid; LPAR: lysophosphatidic acid receptor; lpar1-/-: lpar1 homozygous knockout mouse; LPAR1i/AM966: LPAR1 inhibitor; LY6C: lymphocyte antigen 6 complex, locus C1; LYPLA1: lysophospholipase 1; LYPLA2: lysophospholipase 2; Lyz2: lysozyme 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MARVELD2: MARVEL domain containing 2; 3-MA: 3-methyladenine; MBOAT2: membrane bound O-acyltransferase domain containing 2; MGLL: monoglyceride lipase; MRC1/CD206: mannose receptor C-type 1; MTOR: mechanistic target of rapamycin kinase; NP: normal pregnancy; PDGF: platelet derived growth factor; PLA1A: phospholipase A1 member A; PLA2G4A: phospholipase A2 group IVA; PLPP1: phospholipid phosphatase 1; pMo: peripheral blood monocytes; p-MTOR: phosphorylated MTOR; PPAR: peroxisome proliferator activated receptor; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; PPARGi/GW9662: PPARG inhibitor; PTPRC/CD45: protein tyrosine phosphatase receptor type, C; Rapa: rapamycin; RHEB: Ras homolog, mTORC1 binding; SA: spontaneous abortion; SELE: selectin E; SELL: selectin L; siCLDN7: CLDN7-silenced; STAT: signal transducer and activator of transcription; SQSTM1: sequestosome 1; TJP1: tight junction protein 1; VCAM1: vascular cell adhesion molecule 1; WT: wild type.

Calcineurin inactivation inhibits pyruvate dehydrogenase complex activity and induces the Warburg effect.

Calcineurin is a calcium- and calmodulin-dependent serine/threonine protein phosphatase that connects the Ca2+-dependent signalling to multiple cellular responses. Calcineurin inhibitors (CNIs) have been widely used to suppress immune response in allograft patients. However, CNIs significantly increase cancer incidence in transplant recipients compared with the general population. Accumulating evidence suggests that CNIs may promote the malignant transformation of cancer cells in addition to its role in immunosuppression, but the underlying mechanisms remain poorly understood. Here, we show that calcineurin interacts with pyruvate dehydrogenase complex (PDC), a mitochondrial gatekeeper enzyme that connects two key metabolic pathways of cells, glycolysis and the tricarboxylic acid cycle. Mitochondrial-localized calcineurin dephosphorylates PDHA1 at Ser232, Ser293 and Ser300, and thus enhances PDC enzymatic activity, remodels cellular glycolysis and oxidative phosphorylation, and suppresses cancer cell proliferation. Hypoxia attenuates mitochondrial translocation of calcineurin to promote PDC inactivation. Moreover, CNIs promote metabolic remodelling and the Warburg effect by blocking calcineurin-mediated PDC activation in cancer cells. Our findings indicate that calcineurin is a critical regulator of mitochondrial metabolism and suggest that CNIs may promote tumorigenesis through inhibition of the calcineurin-PDC pathway.

Homocysteine inhibits pro-insulin receptor cleavage and causes insulin resistance via protein cysteine-homocysteinylation.

Elevation in homocysteine (Hcy) level is associated with insulin resistance; however, the causality between them and the underlying mechanism remain elusive. Here, we show that Hcy induces insulin resistance and causes diabetic phenotypes by protein cysteine-homocysteinylation (C-Hcy) of the pro-insulin receptor (pro-IR). Mechanistically, Hcy reacts and modifies cysteine-825 of pro-IR in the endoplasmic reticulum (ER) and abrogates the formation of the original disulfide bond. C-Hcy impairs the interaction between pro-IR and the Furin protease in the Golgi apparatus, thereby hindering the cleavage of pro-IR. In mice, an increase in Hcy level decreases the mature IR level in various tissues, thereby inducing insulin resistance and the type 2 diabetes phenotype. Furthermore, inhibition of C-Hcy in vivo and in vitro by overexpressing protein disulfide isomerase rescues the Hcy-induced phenotypes. In conclusion, C-Hcy in the ER can serve as a potential pharmacological target for developing drugs to prevent insulin resistance and increase insulin sensitivity.